Genetic modification is the process of deliberately creating changes in the genetic material of living cells. Often the purpose is to modify a genetically encoded biological property of that cell, or of the organism of which the cell forms part or into which it can regenerate. These changes can take the form of deletion of parts of the genetic material, addition of exogenous genetic material, or changes in the existing nucleotide sequence of the genetic material, for example by substituting one nucleotide for another.
Methods for the genetic modification of eukaryotic organisms have been known for over 20 years, and have found widespread application in plant, human and animal cells and microorganisms for improvements in the fields of agriculture, human health, food quality and environmental protection.
A common genetic modification methodology consists of adding exogenous DNA fragments to the genome of a cell, which may then confer a new property to that cell or its organism over and above the properties encoded by already existing genes (including applications in which the expression of existing genes will thereby be suppressed).
Although these methods may have some effectiveness in providing the desired properties to a target, these methods are nevertheless not very precise. There is, for example, no control over the genomic positions in which the exogenous DNA fragments are inserted (and hence over the ultimate levels of expression). In addition, the desired effect will have to manifest itself over the natural properties encoded by the original and well-balanced genome. On the contrary, methods of genetic modification that will result in the addition, deletion or conversion of nucleotides in predefined genomic loci will allow the precise and controllable modification of existing genes.
Oligonucleotide-directed Targeted Nucleotide Exchange (TNE) is a method that is based on the delivery into the eukaryotic cell of (synthetic) oligonucleotides (molecules consisting of short stretches of nucleotides and/or nucleotide-like moieties that resemble DNA in their Watson-Crick base pairing properties, but may be chemically different from DNA; (Alexeev and Yoon, 1998); (Rice et al., 2001); (Kmiec, 2003)).
By deliberately designing a mismatch nucleotide in the homology sequence of the oligonucleotide, the mismatch nucleotide may induce changes in the genomic DNA sequence to which the nucleotide may hybridize. This method allows the conversion of one or more nucleotides in the target, and may, for example, be applied to create stop codons in existing genes, resulting in a disruption of their function, or to create codon changes, resulting in genes encoding proteins with altered amino acid composition (protein engineering).
Targeted nucleotide exchange (TNE) has been described in many organisms including plant, animal and yeast cells and is also referred to as Oligonucleotide-directed Mutagenesis (ODM).
The first examples of TNE using chimeric DNA:RNA oligonucleotides came from animal cells (reviewed in (Igoucheva et al., 2001)). TNE using chimeric DNA:RNA oligonucleotides has also been demonstrated in plant cells (Beetham et al., 1999; Kochevenko and Willmitzer, 2003; Okuzaki and Toriyama, 2004; Zhu et al., 2000; Zhu et al., 1999). In general, the frequencies reported in both plant and animal studies were too low for practical application of TNE on non-selectable chromosomal loci. TNE using chimeric oligonucleotides was also found to be difficult to reproduce (Ruiter et al., 2003), resulting in a search for alternative oligonucleotide designs giving more reliable results.
Several laboratories have focused on the use of single stranded (ss) oligonucleotides for TNE. These have been found to give more reproducible results in both plant and animal cells (Liu et al., 2002) (Parekh-Olmedo et al., 2005) (Dong et al., 2006). However, the greatest problem facing the application of TNE in cells of, in particular, higher organisms such as plants remains the relative low efficiency that has been reported so far. In maize a conversion frequency of 1×10−4 has been reported (Zhu et al., 2000). Subsequent studies in tobacco (Kochevenko and Willmitzer, 2003) and rice (Okuzaki and Toriyama, 2004) have reported frequencies of 1×10−6 and 1×10−4 respectively.
TNE using various types of oligonucleotides has been the subject of various patent and patent applications including U.S. Pat. No. 6,936,467, U.S. Pat. No. 7,226,785, US579597, U.S. Pat. No. 6,136,601, US2003/0163849, US2003/0236208, WO03/013226, U.S. Pat. No. 5,594,121 and WO01/92512.
In U.S. Pat. No. 6,936,467 it is contemplated that the low efficiency of gene alteration obtained using unmodified DNA oligonucleotides is the result of degradation of the donor oligonucleotides by nucleases present in the reaction mixture or the target cell. It is proposed to incorporate modified nucleotides that render the resulting oligonucleotides (more) resistant against nucleases. These modifications are disclosed to preferably be located at the ends of the oligonucleotide whereas the mismatch is present at least 8 nucleotides from each terminal end.
U.S. Pat. No. 7,226,785 also discloses methods for targeted chromosomal genomic alterations using modified single-stranded oligonucleotides with at least one modified nuclease-resistant terminal region. TNE using modified single stranded oligonucleotides is also the subject of WO02/26967.
Because of the low efficiency of the current methods of TNE there remains a need for alternative and/or better TNE techniques. These can be used alone or in combination with existing TNE techniques, like those disclosed above and in the art, to improve efficiency. Accordingly, the present inventors have set out to improve on the existing TNE technology.